US4762784A - Fermemtation process for the high level production of bovine growth hormone - Google Patents

Fermemtation process for the high level production of bovine growth hormone Download PDF

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US4762784A
US4762784A US06/754,578 US75457885A US4762784A US 4762784 A US4762784 A US 4762784A US 75457885 A US75457885 A US 75457885A US 4762784 A US4762784 A US 4762784A
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fermentation medium
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Paula M. Keith
Wendy J. Cain
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International Minerals and Chemical Corp
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International Minerals and Chemical Corp
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Assigned to INTERNATIONAL MINERALS & CHEMICAL CORPORATION reassignment INTERNATIONAL MINERALS & CHEMICAL CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: CAIN, WENDY J., KEITH, PAULA M.
Priority to US06/754,578 priority Critical patent/US4762784A/en
Priority to IE187786A priority patent/IE61272B1/en
Priority to IL79404A priority patent/IL79404A0/xx
Priority to AU60200/86A priority patent/AU601157B2/en
Priority to CA000513717A priority patent/CA1279591C/en
Priority to NZ216834A priority patent/NZ216834A/xx
Priority to EP86305430A priority patent/EP0209355B1/en
Priority to DK336886A priority patent/DK336886A/da
Priority to DE8686305430T priority patent/DE3682840D1/de
Priority to JP61164846A priority patent/JPH0716433B2/ja
Priority to ZA865272A priority patent/ZA865272B/xx
Priority to AT86305430T priority patent/ATE70303T1/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C12N15/73—Expression systems using phage (lambda) regulatory sequences
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormones [GH] (Somatotropin)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/818—Aeration or oxygen transfer technique

Definitions

  • This invention relates to high level microbial production of bovine growth hormone through recombinant DNA technology. This high level production is achieved through high-density fermentation of E. coli cells transformed with a recombinant vector carrying a gene encoding bovine growth hormone.
  • Bovine growth hormone is a protein of 191 amino acids, which is initially synthesized in the anterior pituitary land as a precursor "pre-growth hormone” having 26 additional amino acids attached at the N-terminus. This 26-amino acid "signal sequence" is processed off during secretion from the pituitary cells, yielding the mature hormone.
  • Field trials using BGH purified from pituitary glands demonstrated increased milk production and improved feed-to-milk conversion in cows to which the hormone was administered (Machlin, L. J., Journal of Dairy Science, 56:575-580 [1973]). The potential economic value of this hormone sparked interest in obtaining BGH in commercial quantities at reasonable cost.
  • E. coli cells were transformed with the BGH expression vector and BGH synthesis was regulated by the plasmid-borne E. coli trp regulatory region. It is reported that high density fermentation of the transformed E. coli cells yielded approximately 1.5 grams/liter BGH, but no description of the fermentation conditions is given.
  • the present invention provides a method of producing BGH at high levels by fermentation of E. coli cells transformed with a recombinant vector containing a BGH-encoding gene under conditions which optimize the yield of BGH.
  • BGH expression is regulated by a temperature-sesitive repressor encoded by a second plasmid which has also transformed the E. coli host strain.
  • This method of producing BGH comprises inoculating an aqueous fermentation medium with a transformant E. coli strain containing an expression vector which directs the expression of bovine growth hormone under the control of a phage lambda promoter-operator and an expression vector which directs the expression of the ⁇ cI857 temperature-sensitive repressor protein.
  • the transformant strain is grown in the fermentation medium for an initial growth period during which the level of dissolved oxygen in the medium is maintained at from about 20% to 60% of saturation and the temperature of the medium is maintained at between about 26° C. and 30° C. This initial growth period is followed by an induction period during which BGH synthesis is induced by raising the fermentation medium temperature to at least about 42° C.
  • FIG. 1 is a representation of the salient features of plasmid P L -mu- ⁇ 9 (Ser) BGH, a BGH expression vector which can be used in the method of the invention.
  • FIG. 2 is a representation of the salient features of plasmid pcI857, which encodes a temperature-sensitive repressor used to control BGH production in the method of the invention.
  • the plasmid which directs BGH expression in the method of the invention can be any suitable BGH-encoding plasmid in which BGH expression is directed by a regulatory region comprising a promoter-operator region derived from bacteriophage ⁇ , preferably the ⁇ P L promoter-operator region.
  • the regulatory region also contains a Shine-Dalgarno (ribosomal binding) region, which is preferably derived from bacteriophage mu.
  • the BGH-encoding sequence which is operably fused to the regulatory region, comprises a DNA sequence encoding a polypeptide having the amino acid sequence of BGH or a biologically active fragment or analog thereof.
  • BGH bovine growth hormone
  • BGH include fragments of the hormone which may, for example, have varying portions of the amino terminal end of the hormone deleted, or may have various substitutions or modifications in the BGH sequence which do not destroy the biological activity of the polypeptide.
  • BGH polypeptides lacking various portions of the amino terminal end of the hormone have been shown to retain biological activity.
  • the BGH-encoding plasmid encodes ⁇ 9 BGH, i.e., a polypeptide corresponding in amino acid sequence to BGH less the first nine amino-terminal amino acids of the mature hormone.
  • the plasmid also carries a gene encoding a selectable marker, e.g., an antibiotic resistance gene, for selection of cells transformed by the plasmid.
  • a selectable marker e.g., an antibiotic resistance gene
  • the transformant strain employed in the method of the invention also contains a ⁇ cI857 repressor gene.
  • the repressor protein encoded by this temperature-sensitive mutant gene is known to interact with the operators of phage ⁇ gene regulatory regions (including the P L operator) to prevent transcription of genes off the promoter in the regulatory region.
  • This repressor protein has been used to regulate synthesis of desired proteins encoded by recombinant vectors in various transformant strains.
  • C. Queen J. of Molec. and Appl. Genetics, 2:1 1983
  • H. Kupper European Patent Application Publication No. 0 076 037
  • G. Buell European Patent Application Publication No. 0 103 395 All describe the use of the cI857 repressor to regulate synthesis of a recombinant vector-encoded desired protein.
  • the cI857 gene is either carried on the vector carrying the gene for the desired protein (and the ⁇ promoter-operator region directing its expression) or on a separate plasmid transformed into the host cells.
  • Synthesis of the desired protein was repressed by cultivating the transformant host cells at temperatures between 28° C. and 32° C. until the desired cell density was reached. These investigators then inactivated the cI857 repressor (thus inducing synthesis of the desired protein) by raising the temperature to 42°-43° C. for the remainder of the cultivation period.
  • the cI857 gene is used in the method of the invention to control BGH synthesis, and may be carried in the host cell chromosome, on the BGH-encoding plasmid, or on a second plasmid.
  • a second plasmid which directs expression of the cI857 repressor protein is transformed into the host strain along with the BGH-encoding plasmid.
  • the host cells may be any transformable E. coli strain suitable for high density fermentation and in which the expression vectors used to transform the cells will be stably maintained. Many such strains are known in the art, with one suitable strain being E. coli HB101 (Leu Lac pro thi hrs hsm supE recA sm r ).
  • a preferred transformant strain for use in the method of the invention is E. coli HB101 (P L -mu- ⁇ 9 (Ser) BGH and pcI857). Construction of an E. coli transformant containing these plasmids is described in European Patent Application Publication No. 0 103 395, hereinafter referred to as EPO 0 103 395, the disclosure of which is incorporated herein by reference.
  • E. coli HB101 (P L -mu- ⁇ 9 (Ser) BGH and pcI857) has been deposited, with the designation E. coli, IMC No. 1, at the American Type Culture Collection, Rockville, Md., with accession no. 53030. It will be appreciated, however, that the method of the invention is equally applicable to obtain high level production of BGH using other transformant strains in which BGH expression is under control of the cI857 gene product.
  • Plasmid P L -mu- ⁇ 9 (Ser) BGH represented in FIG. 1, encodes a BGH polypeptide lacking the first nine amino-terminal amino acids of the mature hormone and containing an additional serine residue, not normally present in BGH, at the N-terminus.
  • the additional serine residue is present as an artifvact of genetic manipulation at the 5' end of the gene.
  • Expression of the BGH-encoding sequence is controlled by a regulatory region comprising the phage ⁇ P L promoter-operator, a Shine-Dalgarno region derived from bacteriophage mu, and an initiation codon (ATG) adjacent (and 5') to the BGH sequence.
  • the plasmid also carries a gene for ampicillin resistance.
  • the ⁇ 9 (Ser) BGH gene was cloned on plasmid pPLC24 (Gene, 15:81-93, 1981) which is a derivative of pBR322 (G. Sutcliffe, Cold Spring Harbor Symposia, 1978).
  • Point A on the plasmid is nucleotide 4180 in the Sutcliffe sequence.
  • Plasmid pBR322 then continues counterclockwise to the BamHI recognition site at nucleotide 375 of the pBR322 sequence. Clockwise from point A is a 301 base pair fragment from Tn903 which was inserted with the 291 base pair pL promoter.
  • An EcoRI restriction site divides the promoter from a mu sequence which supplies the ribosome binding site up to the initiating ATG codon.
  • DNA is included which codes for serine followed by amino acids 10 through 191, the final amino acid of BGH. This is followed by 65 base pairs of untranslated DNA, 23 dG/dC base pairs from the homopolymeric tails annealed during the original cloning procedure and finally the BamHI recognition site, added with synthetic DNA.
  • Plasmid pcI857 shown in FIG. 2, is a multicopy plasmid which encodes the cI857 temperature-sensitive repressor and also carries a kanamycin resistance gene.
  • E. coli HB101 cells transformed with both plasmids were selected by growth in Luria broth supplemented with both ampicillin and kanamycin by a procedure similar to that described in EPO 0 103 395.
  • the transformant strain is used to inoculate an aqueous medium contained in a fermentor.
  • the aqueous fermentation medium can be any medium suitable for supporting high density growth of E. coli.
  • the medium contains a carbon source, a nitrogen source, salts, and any other nutrients required for cell growth.
  • Suitable carbon sources include, among others, glycerol and hydrated glucose (available commercially as Cerelose®).
  • Suitable nitrogen sources include, among others, acid hydrolysates of casein (commercially available as HyCase Amino Acids or Casamino Acids); enzymatic hydrolysates of casein (NZ Amine A, Casatone, Tryptone); vegetable derived hydrolyzed proteins (soybean peptones, hydrolyzed corn gluten, cottonseed peptone); meat peptones; and yeast extracts.
  • acid hydrolysates of casein commercially available as HyCase Amino Acids or Casamino Acids
  • enzymatic hydrolysates of casein NZ Amine A, Casatone, Tryptone
  • vegetable derived hydrolyzed proteins soybean peptones, hydrolyzed corn gluten, cottonseed peptone
  • meat peptones and yeast extracts.
  • yeast extracts yeast extracts.
  • Any components required for retention of plasmids by host cells are added to the medium.
  • the antibiotics ampicillin and kanamycin are added when the
  • Any conventional fermentation equipment known in the art can be used, provided there are means of controlling the medium temperature, of agitating and aerating the medium, and of adding oxygen to the intake air.
  • the fermentor is inoculated with a culture of the transformant strain.
  • the culture will have been previously incubated at about 30° C. for between 8 and 24 hours (or until the A 550 , i.e., the absorbance at 550 nanometers, of the culture is between 4 and 10) with agitation, for example, at 200 rpm.
  • the culture is incubated at 30° C. for about 15 to 20 hours, or until the A 550 is between 4 and 6.
  • the culture can be grown in any suitable medium, for example, Luria broth.
  • the volume of culture used to inoculate the fermentor is between 1/50th and 1/20th, preferably about 1/25th of the volume of medium contained in the fermentor.
  • the fermentation is conducted in two phases. Following inoculation of the fermentation medium with the transformant strain, an initial growth period is conducted during which the level of dissolved oxygen in the medium is maintained at from 20% to 60% saturation, preferably at about 50% saturation. This may be accomplished by feeding ambient air into the fermentor at a rate sufficient to maintain the dissolved oxygen concentration at the desired level, while also agitating the fermentation medium by any suitable mechanical means. Feeding ambient air at a rate of 0.8 to 1.2, preferably about 1.0, volume of air (STP) per volume of liquid per minute with agitation at 800 to 1200 rpm, preferably about 1000 rpm, is suitable.
  • STP volume of air
  • the agitator is driven by a motor which preferably provides a power input of about 0.5 to 2.0 horsepower per 100 gallons of fermentation medium.
  • the temperature of the medium during the initial growth period is any temperature at which E. coli growth is supported while the cI857 repressor protein is active and BGH expression in the transformant strain is therefore repressed.
  • the temperature is preferably held between 26° C. and 30° C., most preferably at about 28° C.
  • the initial growth period is continued until cell density (as measured by the A 550 of a sample of culture from the fermentor) reaches 50 to 60, which commonly occurs at about 23 to 25 hours after inoculation of the fermentation medium.
  • the second fermentation phase an induction period, is begun.
  • the temperature of the fermentation medium is raised to at least about 42° C. (preferably 42° C.) and held there for about one hour, thereby inactivating the cI857 repressor protein and inducing production of BGH in the transformant strain.
  • the temperature is then reduced to about 38° C. to 41° C., preferably about 40° C. At this temperature, the cI857 repressor protein is inactive but conditions are more favorable for E. coli growth than at 42° C.
  • the dissolved oxygen level in the medium is maintained at from about 10% to 40% of saturation during the induction period. Any suitable means of aeration and agitation can be used to maintain this dissolved oxygen level.
  • ambient air is fed at a rate of 0.8 to 1.2, preferably about 1.0, volumes of air (STP) per volume of liquid per minute, and the medium is agitated at 800 to 1200 rpm, preferably about 1200 rpm.
  • the agitator is driven by a motor which preferably provides a power input of about 0.5 to 2.0 horsepower per 100 gallons of fermentation medium.
  • oxygen present in the ambient air source is fed oxygen into the fermentor in order to maintain the desired dissolved oxygen level.
  • Any conventional means of providing oxygen to the fermentation medium may be employed.
  • a sparger which is connected to an oxygen source may be inserted directly into the medium or oxygen may be added to the ambient air being fed into the fermentor.
  • the induction period is continued until cell density reaches an A 550 of about 80 to 125, preferably 100 to 123. These cell densities are commonly reached at about 7 to 8 hours after the start of the induction period. Fermentation parameters indicating that BGH synthesis and cell growth are complete include: (1) a significant decrease in oxygen demand (2) no further increase in cell density (A 550 values) and (3) NaOH utilization (for pH control) stops.
  • Nutrients which are depleted from the fermentation medium during cell growth are replenished by any of the methods known in the art. Nutrients may be fed continually or in portions during the fermentation. Preferably, nutrients are added in portions three times during the fermentation: when the cell density reaches an A 550 of 30-35, when cell density reaches an A 550 of 50-60, and again at an A 550 of 90-100.
  • the first feeding of nutrients takes place during the initial growth period, usually about 16 hours after inoculation.
  • the second feeding takes place just before the temperature is raised to begin the induction period, usually about 23 to 25 hours after inoculation.
  • the third feeding is given during the induction period, usually about 29 hours after inoculation.
  • the nutrients to be added will depend on the composition of the fermentation medium chosen, but will generally include a carbon source and a nitrogen source.
  • the feedings comprise about equal amounts by weight of NZ Amine A and glycerol.
  • each of the first two feedings comprises a total of about 45-60 grams of the combined nutrients per liter of medium in the fermentor and the third feeding comprises a total of about 20-25 grams of the combined nutrients per liter of medium.
  • We achieved excellent results by adding 250 grams each of NZ Amine A and glycerol in one liter of water to 9.4 liters of fermentation medium at 16 hours post-inoculation and adding another 250 grams each of NZ Amine A and glycerol in one liter of water to the fermentation medium at 24 hours post-inoculation.
  • the BGH produced by the transformant strain may be recovered by any suitable means known in the art.
  • Cells may be harvested from the fermentation medium by, for example, centrifugation. Cells are then lysed by enzymatic, chemical or mechanical means, for example, using sonication, a French press, or treatment with such agents as lysozyme and detergents such as Triton-X-100.
  • BGH may be purified from the cell lysate by any suitable protein purification method, including affinity chromatography, selective precipitation from a solution of a salt such as ammonium sulfate, ion exchange chromatography, isoelectric focusing, or any combination of methods.
  • the fermentation process of the invention has yielded 3.6 to 5.9 grams per liter of ⁇ 9 (Ser) BGH in high density fermentations.
  • Investigators who previously have worked with E. coli hosts transformed with P L -mu- ⁇ 9 (Ser) BGH and pcI857 reported yields of 100 mg/liter ⁇ 9 BGH or less from small cultures, as measured by radioimmunoassay (see EPO 0 103 395).
  • EPO 0 103 395 radioimmunoassay
  • E. coli HB101 P L -mu- ⁇ 9 (Ser) BGH and pcI857 cells, ATCC 53030, to which 10% (v/v) glycerol had been added, were stored under liquid nitrogen or at -85° C. until needed.
  • the inoculum for a 9-liter fermentor charge was obtained by adding the cells to duplicate 500 ml baffled flasks each containing 200 mL of LB medium.
  • the LB medium had the following composition: 10 g per liter tryptone, 5 g per liter yeast extract, 10 g per liter NaCl, 100 ⁇ g/ml ampicillin plus 50 ⁇ g/ml kanamycin.
  • the pH of the medium was adjusted to a value of 7.0.
  • the flasks were closed with a milk filter closure so that some aeration of the medium could take place while the flasks were shaken at 200 rpm for 15-20 hours at 30° C. in a New Brunswick shaker (until the A 550 reached 4-6).
  • composition of the initial 9 liters of medium is shown below:
  • ampicillin and kanamycin were added in sufficient amount to give a concentration of 25 mg/L for each antibiotic.
  • the solution of antibiotics was sterilized by filtration.
  • the fermentor was again fed 250 g NZ Amine A plus 250 g glycerol and the bacteria were induced to synthesize BGH by raising the temperature to 42° C. for one hour.
  • a final feeding of 125 g NZ Amine A plus 125 g glycerol was added so that nutrients were available for the remaining induction period.
  • DO Dissolved oxygen
  • Time Period 0-24 Hours
  • Dissolved oxygen 10-40% of saturation.
  • the inlet air is enriched with oxygen and mixed prior to introduction to the fermentor through a sparger.
  • fermentor broth samples were collected by centrifugation (10-15,000 ⁇ g, 15 min.) and bacteria were resuspended in 3-5 volumes of buffered guanidine (8M guanidine HCl, 50 mM glycine NaOH buffer, pH 9.8, 5 mM reduced glutathione). The suspension was allowed to sit for 20-30 min. and was then homogenized (15-20 seconds) with a model SDT-1810 Tek-Mar tissue mizer. Insoluble debris was removed by centrifugation as above and the clarified BGH extract was assayed by HPLC.
  • buffered guanidine 8M guanidine HCl, 50 mM glycine NaOH buffer, pH 9.8, 5 mM reduced glutathione

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US06/754,578 1985-07-15 1985-07-15 Fermemtation process for the high level production of bovine growth hormone Expired - Fee Related US4762784A (en)

Priority Applications (12)

Application Number Priority Date Filing Date Title
US06/754,578 US4762784A (en) 1985-07-15 1985-07-15 Fermemtation process for the high level production of bovine growth hormone
IE187786A IE61272B1 (en) 1985-07-15 1986-07-14 High level production of bovine growth hormone
IL79404A IL79404A0 (en) 1985-07-15 1986-07-14 High level production of bovine growth hormone
AU60200/86A AU601157B2 (en) 1985-07-15 1986-07-14 High level production of bovine growth hormone
CA000513717A CA1279591C (en) 1985-07-15 1986-07-14 High level production of bovine growth hormone
NZ216834A NZ216834A (en) 1985-07-15 1986-07-14 Production of bovine growth hormone by use of recombinant dna
EP86305430A EP0209355B1 (en) 1985-07-15 1986-07-15 High level microbial production of bovine growth hormone
DK336886A DK336886A (da) 1985-07-15 1986-07-15 Storproduktion af bovint vaeksthormon
DE8686305430T DE3682840D1 (de) 1985-07-15 1986-07-15 Mikrobielle herstellung von rinderwachstumshormon in grossem ausmass.
JP61164846A JPH0716433B2 (ja) 1985-07-15 1986-07-15 ウシ成長ホルモンの高レベル産生
ZA865272A ZA865272B (en) 1985-07-15 1986-07-15 High level production of bovine growth hormones
AT86305430T ATE70303T1 (de) 1985-07-15 1986-07-15 Mikrobielle herstellung von rinderwachstumshormon in grossem ausmass.

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AU (1) AU601157B2 (da)
CA (1) CA1279591C (da)
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DK (1) DK336886A (da)
IE (1) IE61272B1 (da)
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US5104796A (en) * 1987-04-06 1992-04-14 International Minerals & Chemical Corp. High titer production of human somatomedin c
US5342763A (en) * 1992-11-23 1994-08-30 Genentech, Inc. Method for producing polypeptide via bacterial fermentation
CN101591690B (zh) * 2008-05-29 2012-02-01 北京凯因科技股份有限公司 一种重组人干扰素α2b的高密度发酵方法
WO2017192925A1 (en) * 2016-05-05 2017-11-09 William Marsh Rice University Improved microbial production of fats

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US4788144A (en) * 1985-06-28 1988-11-29 International Minerals & Chemical Corp. Fermentation process for the high level production of swine growth
US5622845A (en) * 1988-02-17 1997-04-22 The Upjohn Company Fermentation method for producing norleucine
DE68927997T2 (de) * 1988-02-17 1997-09-11 Upjohn Co Verfahren zum regeln des norleucingehalts in polypeptiden
CN104593318B (zh) * 2013-10-31 2018-05-04 中国食品发酵工业研究院 一种用于细胞培养基的玉米活性肽添加剂
US9534026B2 (en) 2013-10-31 2017-01-03 China National Research Institute Of Food & Fermentation Industries Corn active peptide additive for cell culture medium

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EP0111814A3 (en) * 1982-12-16 1985-08-14 Mgi Pharma, Inc. The cloning and expression of nucleotide sequences encoding bovine growth hormone
BG49718A3 (en) * 1983-07-15 1992-01-15 Bio Technology General Corp Method for preparing of polypeptid with superoxiddismutasne activitty

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US4167450A (en) * 1977-07-13 1979-09-11 University Of New Hampshire Method and apparatus for the production of secondary metabolites by the maintenance-state cultivation of microorganisms
US4317882A (en) * 1979-04-26 1982-03-02 Asahi Kasei Kogyo Kabushiki Kaisha Production of plasminogen activator
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US5104796A (en) * 1987-04-06 1992-04-14 International Minerals & Chemical Corp. High titer production of human somatomedin c
US5342763A (en) * 1992-11-23 1994-08-30 Genentech, Inc. Method for producing polypeptide via bacterial fermentation
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US5633165A (en) * 1992-11-23 1997-05-27 Genentech, Inc. Fermentor with vertical shaft
CN101591690B (zh) * 2008-05-29 2012-02-01 北京凯因科技股份有限公司 一种重组人干扰素α2b的高密度发酵方法
WO2017192925A1 (en) * 2016-05-05 2017-11-09 William Marsh Rice University Improved microbial production of fats
US10920251B2 (en) 2016-05-05 2021-02-16 William Marsh Rice University Microbial production of fats

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CA1279591C (en) 1991-01-29
EP0209355B1 (en) 1991-12-11
IL79404A0 (en) 1986-10-31
JPH0716433B2 (ja) 1995-03-01
EP0209355A1 (en) 1987-01-21
IE61272B1 (en) 1994-10-19
DK336886D0 (da) 1986-07-15
NZ216834A (en) 1988-08-30
ATE70303T1 (de) 1991-12-15
ZA865272B (en) 1987-03-25
AU601157B2 (en) 1990-09-06
AU6020086A (en) 1987-01-22
JPS6265698A (ja) 1987-03-24
DK336886A (da) 1987-01-16
IE861877L (en) 1987-01-15
DE3682840D1 (de) 1992-01-23

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